Mitochondrial Oxidative Stress in the Retinal Pigment Epithelium as a Model for A

描述（由申请人提供）：年龄相关性黄斑变性（AMD）是我国老年人的主要致盲疾病，虽然VEGF抑制剂经常阻止疾病的晚期，分泌期，但对于早期萎缩性AMD没有有效的治疗方法。视网膜色素上皮（RPE）的氧化损伤通过降低这些细胞的功能和刺激炎症级联导致疾病的病理特征，视网膜局部萎缩和脉络膜新生血管，从而导致AMD。损伤RPE的活性氧（ROS）的一个来源是RPE细胞本身的线粒体。为了确定线粒体是否是体内RPE中ROS的重要来源，我们将在小鼠RPE中特异性地删除编码保护酶MnSOD的SOD2基因，使用含有SOD2等位基因的小鼠细胞系和RPE特异性表达的Cre重组酶的loxP位点。我们计划进行3组实验：(1)利用电生理（ERG）、高分辨率结构分析（SD-OCT）和行为分析（Optomotry）实时监测活体小鼠视网膜变性的时间过程。在死后样本中，我们将检查RPE、布鲁赫膜和神经视网膜的形态学变化，并测量RPE中脂褐素的积累。我们将在新生小鼠和成年小鼠中诱导SOD2的缺失，以确定哪种方法更好地模拟人类地理萎缩。(2)在转录因子Nrl基因纯合突变的小鼠中，我们将通过删除SOD2来增加线粒体氧化应激。这些老鼠有一个只有锥体的视网膜，可能比正常老鼠的视网膜更像富含锥体的人类黄斑。通过这种方法，我们希望了解为什么中央视网膜比周围视网膜对氧化应激更敏感。(3)我们将尝试通过提高一组抗氧化酶，包括血红素加氧酶-1、谷胱甘肽转移酶和NAD(P)H：醌氧化还原酶1来对抗线粒体氧化应激。这些酶的基因包含一个共同的序列成分，表示抗氧化序列元件“ARE”。我们将通过两种方式刺激这种抗氧化反应——通过病毒传递一种基因来刺激这种反应，以及通过口服一种新药来激活这种途径。我们希望这些方法可能导致萎缩性AMD的治疗。

DESCRIPTION (provided by applicant): Age Related Macular Degeneration (AMD) is a major blinding disease of the elderly in this country and, while VEGF inhibitors often stem the late, exudative stages of the disease, there is no effective therapy for the earlier, atrophic form of AMD. Oxidative damage to the retinal pigment epithelium (RPE) contributes to AMD by reducing the function of these cells and by stimulating an inflammatory cascade that leads to the pathologic hallmarks of the disease, localized atrophy of the retina and choroidal neovascularization. One source of the RPE- damaging reactive oxygen species (ROS) are the mitochondria of the RPE cells themselves. To determine whether mitochondria are an important source of ROS in the RPE in vivo, the SOD2 gene, encoding the protective enzyme MnSOD, will be deleted specifically in the RPE of mice, using a mouse line containing an allele of SOD2 flanked by loxP sites and RPE-specific expression of Cre recombinase. We plan 3 sets of experiments: (1) We will monitor the time course of retinal degeneration in real time in living mice using electrophysiology (ERG), high resolution structural analysis (SD-OCT) and behavioral analysis (Optomotry). In post mortem samples, we will examine morphological changes to the RPE, Bruch's membrane and the neural retina and to measure accumulation of lipofuscin in the RPE. We will induce the deletion of SOD2 both in neonatal and in adult mice to determine which approach better models human geographic atrophy. (2) We will increase mitochondrial oxidative stress by deletion of SOD2 in mice bearing a homozygous mutation in the gene for the transcription factor Nrl. These mice have a cone-only retina which may resemble the cone-rich human macula better than the normal mouse retina. By this means we hope to learn why the central retina is more sensitive to oxidative stress than the peripheral retina. (3) We will attempt to counteract mitochondrial oxidative stress by elevating a set of antioxidant enzymes, including heme oxygenase-1, glutathione transferases, and NAD(P)H:quinone oxidoreductase 1. These genes for these enzymes contain a common sequence component denoted "ARE" for antioxidant sequence element. We will stimulate this antioxidant response in two ways-by viral delivery of a gene that stimulates the response and by oral delivery of a novel drug that activates this pathway. We hope that these approaches may lead to a therapy for the atrophic form of AMD. PUBLIC HEALTH RELEVANCE: Age related macular degeneration (AMD) is the leading cause of blindness among the elderly in the United States. It is caused by degradation of the central retina, an area called the macula. There are two forms of the disease: atrophic or "dry" AMD and exudative or "wet" AMD. This project seeks to establish a mouse model of the pathology related to AMD and to use this model to test potential therapies for the atrophic form of the disease.